Secretion and endocytosis play critical roles in the health and function of both individual cells and multi-cellular organisms to control a wide variety of physiological processes including nutrient uptake, secretion of hormones and digestive enzymes, synaptic transmission between nerve cells, and defense against foreign pathogens. Both secretion and endocytosis critically depend on the transport of cargo, e.g., polypeptide hormones, between various intracellular organelles of these pathways. One mechanism for transporting cargo involves the formation of small membranous vesicles that bud from one compartment and travel to and fuse with a target compartment. More recently, however, another possible means for transporting material between intracellular compartments has become the subject of growing interest-namely, the formation of uniformly sized membrane tubules from the Golgi complex, trans Golgi network (TGN), and endosomes. Little is known about the exact function of these tubules, or how they are formed. Over the last two years of this grant, we have been performing basic cell biological research in order to elucidate the function of these tubules and their molecular mechanism of formation in mammalian cells. To do this, we have developed and utilized both in vitro and permeabilized cell systems which reconstitute tubule formation from both Golgi and endosome membranes. Using these, and other in vivo assays, we have obtained complementary lines of evidence indicating that membrane tubulation uniquely requires a cytoplasmic Ca2+- independent phospholipase A2 (PLA2) enzyme. The central hypothesis that we propose to test is that a specific cytoplasmic PLA2 is intimately involved in the formation of membrane tubules that function in Golgi-to-ER retrograde trafficking, and the assembly of an intact, fully interconnected Golgi complex. Our plan is to identify the specific enzyme, isolate a cDNA, and prepare antibodies against the protein. These reagents, along with pharmacological inhibitors of PLA2s, will then be used to explore the specific functions of tubules in cells through over- expression and ablation and in in vitro reconstitution assays. These studies will contribute to the elucidation of novel PLA2- dependent, membrane tubule-mediated intracellular trafficking events in the secretory pathway.